Translation of proteins by the ribosome is key aspect of life. Structural studies have provided a three- dimensional view of the 2.5 megaDalton 70S ribosomal particle, and revealed the active sites for decoding of genetic information and peptide bond formation. Mechanistic views have revealed the basic steps of translation: initiation, elongation and termination. However, limited dynamic information about how the translational machinery changes conformation and composition are available. Our research focuses on providing a dynamic view of translation using single-molecule approaches. The current proposal builds on the progress in observing ribosomal dynamics in real time using single- molecule fluorescence approaches. We will monitor the underlying dynamics of translation in four specific aims. First, we will explore the mechanism of initiation, in which the 70S ribosomal particle assembles at the correct start codon; we will use single molecule fluorescence to understand intersubunit joining and dynamics, the role of factors in initiation, and movements of tRNAs, factors and ligands during the process. These experiments will be complemented by single-molecule force measurements to map the energetics of mRNA-ribosome interaction. In aim 2, we will explore the nature of intersubunit conformational changes that underlie both initiation and elongation. We will determine the nature of the barrier to rotation using both fluorescence and force and explore how factor and tRNA dynamics are correlated to ribosomal motions. We will determine the effects of ribosomal mutations and composition, as well as antibiotics, on conformational dynamics. In aim 3, we will apply new single-molecule instrumentation to perform single-molecule experiments under high concentrations (50nM-5M) of labeled tRNAs, ribosomes or factors. These experiments harness zero mode waveguides, and will allow us to observe rapid changes in ribosome composition, and correlate conformational changes with each other. Finally, in aim 4, the dynamics of ribosomal translation will be explored during translation to understand codon and mRNA context effects. We will use intersubunit FRET, tRNA colocalization and ribosome mRNA -FRET to determine dynamics effects on model mRNAs, and then use these systems to explore the dynamics of rare recoding events. The results of this project should reveal a dynamic view of translation and continue to provide insights into the mechanism of action of antibiotics.